Amino acids and their derivatives are important precursors in the pharmaceutical industry and added to a wide variety of food and feed as supplements. Several amino acids, such as glutamate, lysine and threonine are produced using their natural biosynthetic pathways. In natural amino acid biosynthesis the amino acid aspartate serves as the precursor for the production of other amino acids, such as lysine, threonine, isoleucine and methionine.
With a world-market of 900,000 t/a, the essential amino acid L-lysine is one of the most important biotechnological fermentation products (Kohl and Tauch, 2009). It is mainly applied as supplement to animal feed (Anastassiadis, 2007). Supplementation of such feed materials with a lysine rich source leads to optimized growth of e.g. pigs or chicken. The continuing increase in consumption of white meat has led to an increased demand for lysine during the past decades.
Lysine can be produced by e.g. fermentation methods. For this purpose, certain microorganisms such as C. glutamicum have been proven to be particularly suited. Fermentation as technique for the industrial production of amino acids emerged with the discovery of the glutamate secreting bacterium Corynebacterium glutamicum. Within a few years from its discovery, the first lysine excreting mutants of C. glutamicum were applied for large scale production (Kinoshita, et al., 1961). Research continues to be directed towards new technologies to establish high-efficient fermentation including optimization of the fermentation procedure, down-stream processing as well as strain engineering.
Classically strains were engineered by an iterative approach of random mutagenesis with UV light or chemical mutagens and subsequent strain selection. The key to success in these days was the use of toxic lysine analogues, such as S-(2-aminoethyl) cysteine, to select for feedback resistant strains (Nakayama and Araki, 1973). These classical strains typically shared point mutations in the aspartokinase gene, which release the encoded enzyme from feedback inhibition by lysine and threonine (Kalinowski, et al., 1991; Thierbach, et al., 1990). Remarkable production properties such as a conversion yield up to 50% and a lysine•Cl titre of 100 g L−1 are achieved with such classically derived strains (Leuchtenberger, et al., 2005). This was, however, typically linked to extensive fermentation times of 2-3 days, limiting productivity. Additionally, auxotrophy and the weak stress tolerance, resulting from undesired mutations which accumulated during strain development (Ohnishi, et al., 2002), further display severe disadvantages of conventional production strains. In recent years, recombinant DNA technology and molecular biology initiated a new era of strain engineering-rational optimization by metabolic engineering (Ikeda, et al., 2006). Many of these studies have focused on optimization of the flux through the lysine biosynthesis by directly modifying enzymes of this pathway. The release of aspartate kinase from feedback control is today regarded as one of the most important features of industrial production strains. Beside modifications concerning pathway regulation, the intracellular activity of rate determining enzymes of the biosynthetic pathway is a key point for strain engineering. Strategies for increasing enzyme activity within the cell involve overexpression by the use of stronger promoters, mutating the promoter sequences or regulatory regions upstream of the gene, or increasing the copy number of the coding gene. Plasmid-related overexpression in this context is appropriate to achieve higher enzyme activities and better lysine yields (Eggeling, et al., 1998) but can hardly be applied in an industrial process.
Identification of beneficial targets apart from the biosynthetic pathway itself became soon necessary to abolish bottlenecks within the precursor and co-factor supply towards creation of competitive production strains. This is, however, more challenging and difficult as it requires understanding of the organism on a systems level. In this regard, the availability of the genome sequence of Corynebacterium glutamicum has been a mile stone for metabolic engineering (Haberhauer, et al., 2001; Kalinowski, et al., 2003; Ohnishi, et al., 2002; Pompejus, et al., 2002). It provided the basis for (i) genome breeding by comparative sequence analysis between classically derived production strains and the wild type (Ohnishi, et al., 2002), (ii) a detailed in silico reconstruction of the metabolic network of C. glutamicum (Kjeldsen and Nielsen, 2009) including stoichiometric modelling approaches to analyze the theoretical production capacity as well as metabolic pathways involved (Kromer, et al., 2006; Wittmann and Becker, 2007), and (iii) the discovery of transcriptional regulatory networks by means of specific sequence motives within the genome (Kohl and Tauch, 2009). These models, however, are not applicable to predict the activity of the metabolic pathways in vivo, i.e. the fluxome, as key characteristic for systems understanding and guidance of strain engineering. Flux analysis is a central element of metabolic engineering (Stephanopoulos, 1999), as indicated by the impressive progress to estimate metabolic fluxes in vivo (Christensen and Nielsen, 2000; Christensen, et al., 2000; Frick and Wittmann, 2005; Van Dien, et al., 2006; Wittmann, 2007; Wittmann and Heinzle, 2002). Beyond the insight into the biological system, 13C metabolic flux analysis has proven useful for strain characterization and identification of beneficial targets for lysine production (Kiefer, et al., 2004; Wittmann and Heinzle, 2002). Together with complementary findings from determination of the active set of genes (transcriptome) (Hayashi, et al., 2006) and proteins (proteome) (Bendt, et al., 2003) and from quantification of intracellular metabolite levels (metabolome) (Borner, et al., 2007) an extensive data set is provided to gain a deep insight into cell physiology on a global level. This systems-oriented approach displays an excellent platform for metabolic engineering (Lee, et al., 2005).
The major lysine-producing microorganism Corynebacterium glutamicum was discovered in the 1950s in a large screening program in Japan. Strains were successively optimized for lysine production using an iterative process of random mutagenesis and screening for improved production characteristics. This resulted in efficient production strains but also led to an accumulation of side-mutations causing impaired growth, weak stress tolerance or increased nutrition demands. Accordingly, the production performance currently obtained is significantly below the theoretical capacity predicted for C. glutamicum. The progress in molecular biology and systems-oriented tools for the analysis of the metabolism and regulatory state of the cell nowadays shifts strain engineering to a more precise and targeted optimization systems metabolic engineering. This aims at superior hyper-producing strains with exclusive sets of beneficial modifications exhibiting increased production yield, titre and productivity.
In the past, various attempts have been made to increase the production of L-lysine using microorganisms. General attempts to increase production of e.g. methionine or lysine by up- and/or downregulating the expression of genes being involved in the biosynthesis of methionine or lysine are e.g. described in WO 02/10209, WO 2006/008097, and WO 2005/059093.
The central catabolic network, previously identified in C. glutamicum, comprises the pathways of glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle and glyoxylate shunt.
WO 03/042389 relates to a genetically modified microorganism into which a G6PD gene (zwf) has been introduced or in which a G6PD gene has been modified, and the use of such microorganism in preparing a compound of interest, such as an amino acid, particularly preferably lysine.
WO 0220542 relates to a process for the fermentative preparation of L-amino acids, in particular L-lysine, in which the metabolic pathways which reduce the formation of the desired L-amino acid are at least partly eliminated are employed, e.g. using an enhanced gap2 gene. A long list of additional genes that may be modified is mentioned as well.
EP 0435132 discloses microorganisms of the genus Corynebacterium or Brevibacterium which contain a recombinant DNA and are suitable for obtaining amino acids, especially L-lysine. The DNA sequences are derived, in particular, from L-lysine-producing strains of the genus Corynebacterium or Brevibacterium, preferably from a mutant obtained by mutagenesis of Corynebacterium glutamicum ATCC 13032 with reduced feedback inhibition of aspartate kinase.
EP1067193 discloses an L-lysine producing coryneform bacterium (A) with an amplified pyc (pyruvate carboxylase) gene in which at least one of the additional genes dapA (dihydropicolinate synthase), lysC (aspartate kinase), lysE (lysine exportercarrier) and/or dapB (dihydropicolinate reductase) is amplified, preferably over-expressed.
EP0854189 discloses a coryneform bacterium harboring an aspartokinase in which feedback inhibition by L-lysine and L-threonine is substantially desensitized, and comprising an enhanced DNA sequence coding for a dihydrodipicolinate reductase, an enhanced DNA sequence coding for dihydropicolinate reductase, an enhanced DNA sequence coding for dihydropicolinate synthase, an enhanced DNA sequence coding for diaminopimelate decarboxylase and an enhanced DNA sequence coding for aspartate aminotransferase.
EP0857784 relates to a DNA comprising aspartokinase gene free from feed back inhibition by L-lysine, etc., and containing diaminopimelic acid decarboxylase gene and improved in L-lysine-producing ability of a coryneform bacterium.
WO/2007/017526 discloses a process for the preparation of a aspartate and derived amino acids like lysine, threonine, isoleucine, methionine, or homoserine employing microorganism with enhanced isocitrate lyase and/or malate synthase expression.